PL106350B1 - METHOD OF PREPARATION OF 3 DERIVATIVES ALPHA-H-7A BETA-METHYLHYDROGENIDANONE-1 - Google Patents

Description

The patent description was published: September 30, 1980, ( 106350 Int. CL * C07D 311/78 // C12D 13/00 C07C 35/32 READING ROOM Ur-c-du Patent J f t | IzcezttHfifMj Utat | Inventors: John Costain Knight, Merle Gene (Wovcha) Patent holder: The Upjohn Company, Kalamazoo (US the Americas) The method of producing derivatives 3 < * - H-7aP-methyl hexahydroindanone-1 i The subject of the invention is a method for producing derivatives of 3a ^ -7a £ -methylshe ^ owodoxoin- dainon-yl of formula 1, 2, 3 and 4 used as intermediate products for useful products steroids.

Conversion of steroids carried out with the use of microorganisms is widely known and described in literature. One of the earliest opulbflri- works are the publications of Mamoli and Vercello- from 1937 Ber. 70, 470 and Beir. 70, 207 and 9. Au¬ these authors described the 17-keto steroid reduction process to 17-p-hydroxy steroids, carried out with using fermentation yeast. In a later in the work of Patarson and Murray, the process lla-hy- progesterone droxylation, conducted with use Rhizepus ntigricans fungi (patent specification St. United States No. 2,602,709).

Also recently in the 'US patent US

In America 3,684,657, selective microbiota is described the logical process of decomposition of 1.7-alkyloste.roikel, v wherein the 17'alklyl group is at least less 8 carbon atoms carried to the target obtaining andxoist € no-4-dioinu-3 ^ 17, andiroBtadieno- - -1,4-onu-3 using Mycobacterium sp MRRL B-3686 ,,. In an even later patent St. US No. 3,759,791 was selected selectively ne, microbial production of andirosteno-4- -dione of the ^ 3yl7 by fermentation of a steroid from the series cholastan or stigmastane, having, in half 17 alkyl groups of at least 8 atoms carbon, carried out using Mycobacterium sp. NlfcRL B-3806.

The compounds of formula 1 and formula 2 are new.

Admittedly, Schubert et al. described in 1964 (Ste- roid! s 4,5®1— (586) Compound of Formula 1, but it was open ring form, i.e. 7a-methyl- -perhydroindanedione-4-(p-propyl-4) which is rose as an intermediate product of progesterone breakdown with Myoabaoterium smegmatis.

The compound of formula III, produced by the process in the debt of the invention was described in 1963 (Wang and Siih Biochem. 2, 1.23 (8- ^ 1243) as product intermediate decomposition of andirosteno-4-ol- »17p-onlu-3, at using the Necardia restrictus strain.

The method according to the invention is application to decompose! mutant steroids capable of se¬ decomposition of steroids, possibly preventing having at position 17 an alkyl side chain with 2 to 10 carbon aitom and an accumulation in the boundary fermentation tank of the compound of formula 1, i.e. he- mnketalu 3aa-H-4a- / 3, Hnyroxypropyl / -7afjHmety- - hexahydroindanedione Hl.5, a compound of formula 2, i.e. hemiacetal 3aa-H-4a- / 3, -ihydroxypropy- le / -5awhydToxy-7ap- ^ methylsize'sciowcd rhodndanone- -1, a compound of formula 3, i.e. 5-lactone 3act-H-a- / 2'- - carboxyethyl) &lt; 5 &gt; Hhydroxy-7ap-m doroindanone-1 and the compound of formula 4, i.e. 3aa- ^ - ^ V; 3 / ^ ydrc) il ^ ypropyl / -i5a-nydxoxy-7ap- (me- tyloscihydroindainone-1. 106 350106: 3 The mutants used in the method according to the invention You can get the fineness from the following types sterol-degrading organisms when using food the ways of carrying out the mutations and the represented ones further on in the description of various methods of mutation waaiia of microbes: ATtnirobacter, BaciiBus, BTe- vifoacteri! um, Corynebacteriuim, Mycrobacteriuiin, No- cardia, Protaminobacter, Serratia and Streptomyces.

The genus Mycofoacteriutm is preferably used.

Examples of strains of this genus are Myco-10 bacteriuim phlei, Mycobacteriuim smegmatis, Myco¬ bacterium rhodochrous>, Mycobacteriuim miucosum, Mycobacterium fortuitum and Mycobacterium bu- tyrkuim. The method of in invention debt carried out using the new 15 mutant of the Mycobacterium fortudltum NIRRiL strain B-81 & 8, which is used for selective decomposition a large amount of steroids possibly contained in it Side alkyl chain of 2-10 atoms carbon to compounds of formula 1, 2, 3 and 4. Example 20 Some suitable steroids are: sitosterols, cholesterol, stigmasterol, campesterol and the like 17-alkylated steroids side chain with 2 ^ -10 carbon atoms and andro- steno-4'-dione.H3.17, andirostadieno-1,4-d} ion-3y1.7 dehy- droepiaindrosterone and testesterone. Steroid substrates we can be used both in pure form and in raw form.

Mutants characterized by the ability to se decomposing of the above-mentioned helmets and to collect compounds of formula 1, 2, 3, and 4 in the fermentation broth can be used jump by carrying out mutations of microorganisms, capable of breaking down sterols, of the following genera: Antlhrobacter, Bacillus, Brevifoacterium, * Corynabacteriuirn, Mycobacterium, Nocardia, Pro- taiminobacter, Serratia and Streptomyces. Mycofoacte- nium fortuiitum ATCC 684i2, undergoes the process of mutation with each other described in the following part of the description to give a no you, a laboratory mutant microbe. W kata- * 0 the ATCC log from 1974 next to the entry ATCC 6842 za¬ the information "J. C. Cruz 2. Goid aifoscess.

Acta Med. Rio> de Janeiro 1: 1 (1986). Medium 90 37C ". Strain Myicobaicterium fortuitum ATCC 6842 breaks down sterols non-selectively into compounds * with a low molecular weight, for example to carbon dioxide and water. So, fine this regime is unsuitable for selective decomposition a lot of steroids. 50 Mutation of Mycobacterium fortuitum ATCC strain 6842, carried out with the use of nitrosoguanides- ny produces a new mutant which it selectively decomposes the previously mentioned steroids with the preparation compounds of formula L, 2, 3 and 4. This mutant a the microorganism of the Mycobacterium fortuitum strain obtained the NHHRL B-8128 register number issued by Northern Regional Research Laboratory, Mtiinister- of the United States of America, Peoria, Illinois, United States of America, M where this mutant has been deposited in the permanent collection.

Upon request, subculture of this mutant is easy to use I'm from the mentioned collection. Zroztuimiale is, that the availability of breeding does not constitute a permit practicing the method according to the invention 4 and the reduction of patent rights, including the litter threatens to institute court proceedings.

Morphology and susceptibility to drugs of the strain- Myco¬ bacterium NRRL B-S128 does not differ from the responses the characteristics of the parental Mycobacte strain rium fortuitum ATCC 6842. Both discussed strains of Mycobacterium fortulittum do not produce fatty acids, do not produce spores, belongs to the Mycofoaoteriacese family of the Actimo- mycetales. According to the classification of Rumyoma (Ru- nyon E. H., 1959 Med. Clin. North America 43, 273) constitute the lachromogenic group of mycoba cterium IV, that is, these strains grow rapidly at low temperature, giving the colonies a stained on relatively simple media.

Mycobacterium fortuitum ATCC 6842 strains and Mycobacterium fortuiltum NRRL B-αi28, vp does not differ in the action of steroids on molecules.

As described above, the Mycobacteriuim fortui strain ATCC 6-842 tumors steroids non-selectively, while the strain Mycobacteriuim fortuitum MRRL B-8128 breaks down these substances selectively.

This property of the Mycobacterium fortuitum strain NRjRL B- & 128 makes this strain highly useful.

Mutations of the strain Mycofoacteriimi ^ ortuitum ATCC 6842 to obtain the strain - Mycobacterium form tuitum NRRL B 8III 218 is carried out using nitrosoguanidines.

The method of carrying out the mutation in detail described later in the description. Anyway that the mutations are generally known, no the theory is now known to allow, define or even suggest the type of mutant that might be cannot be obtained by using the appropriate how to carry out the mutation. Also, although the method for carrying out mutations and changes the shape is described in detail with reference to strain of Mycobacterium, it is understood that a similar ny or equilibrium course of action- you can to be used in reference to other organisms the types mentioned in the description.

The method according to the invention proceeds with by the aid of a growing culture of the Mycobacte strain rium fortuitum NURL B-8128, adding to the culture during the incubation, the steroid subistrat or b is introduced using steroid suibsitrate directly nutrients before inoculation. The steroid can be added single barrel in conjunction with other controls dem. Preferably, but not limited to, the stereoid is a sto It is used at a concentration of about 0.1 to about 100 g per liter three breeding.

Breeding grows on the medium of growth which as a carbon source, for example, it contains functional hydrocarbons and as a nitrogen source, for example a class of assimilable nitrogen compounds or substances proteinaceous. A table is preferably used as a source are used for glucose, molasses, sugar, glycerin, starches, corn starch, lactose, dextrin, bnu sugar- natoiy and the like. The preferred nitrogen sources are no corn steep extract, yeast jam, autolized brewed yeast with solids substances obtained from milk, soybean milk, cotton grain flour, cornmeal, sub steadily derived from milk, pamkreatine casein extract, fishmeal, distillery sufo-106 350 6 steady state, extract of animal peptone, dropped out in meat and bones, salt. ammonium and the like.

Cut the seeds of nitrogen with manna as you like.

No need to add to the fermentation broth wac..v motali in trace amounts, for example cynic, magmezti, mangatnu, cobalt, iron and the like alych, ppmdewiaz. as ingredients for necklaces, before its wastage * wiajniemv used tap water and uncleaned other ingredients.

The transformation process takes place in the body guo 7) 2 hours to 15 days. During the trial the incubation temperature is kept within limits ° -37 ° C, preferably 30 ° C. The contents of the vessel, in which the transformations in the price are carried out or to facilitate the growth of the microorganism and thus increasing the efficiency of the transformation process, he aerates himself with exhausted air and shakes himself are. After completing the transformation process, oo he states the judgment using thin-film radiographs with the use of gel-coated plates silica (E. Merck, Dairmstadt) and ethyl and cyclohexane in the ratio by volume 2: 3, the converted steroids are isolated by name new way.

For example, the fermentation broth after training, consisting of a fermentation liquid and microbial cells can extract or¬ an organic steroid solvent raging in the water. Dissolve as appropriate the ingredients are methylene chloride (preferably), chloroform, carbon tetrachloride, ethylene chloride, trichlorethylene, ether, amyl acetate, benzene and the like similar.

Alternatively, fermentation broth and cells the fine particles are first separated into ananus person, e.g. by sieving or centrifuging, followed by liquid and thinner are continuously extracted separately with a suitable solvent.

Cells can be extracted either by dissolving with a mixing kettle and do not mix with water. Cell-free liquid fer¬ mentacyin can be extracted with a solvent immiscible with water.

Extracts can be soaked through a layer of earth diatomaceous and completely distilled from the feed draw the solvent under reduced pressure.

Residue containing transformed stereoids, it is then dissolved in a 10% chlorine solution formula in Skellysolve B and chromatographed on silica gel using as eluent Skellysolve B (isomeric hexane mixture) and mixtures thereof with increasing amounts of acetate ethyl. In this way, compounds of a pattern are washed out Rach 1, 2 and 3. The relationship about the vision 4 of the mozina elution for example, with a 5% methanol solution in ethyl acetate. Crystalline products are obtained when used as a solvent, na example of ethyl acetate. Converted steroids can also be obtained after evaporating the dissolves decant from solutions remaining after decant tions.

Compounds of formulas 1, 2, 3 and 4 are used as intermediates in chemical synthesis were obtained useful steroids. For example, listed 40 45 50 59 compounds can be transformed into starting substances partial used in the process described in the description U.S. Patent Eat. No. 3,880,084, pp concerning the way of carrying out total syn theses of useful l ^ Hnorsteroids. .Such a switch shaping into starting materials can be transformed lead in a known way, for example 'via oxidation with chromic acid in acid. vinegar and then exposure to the anhydride acetic acid and sodium vinegar (Biocheim. 2, 1 £ 38— ^ 1243 and J. A. C. S. 85, 2135 ^ 2137).

The method according to the invention and the products twoinzones this way are written more closely in na¬ tap dancing examples. If not specified, more generally, all percentages given in the examples are weight, and all the ratios of scale all solvent mixtures are volumetric.

Example I. Production of the Mycoba mutant oteriiuim fortuitum NRL B-1828 from strain Mycoba- oterium fortuitum ATCC 6842. a) Nitrosoguanidine mutagenesis. Strain cells Mycobacterium fortuitum ATCC 6842 gives up growth at 28 ° C on disalted a culture medium with the following composition: Broth (Di-fco) 8 g / liter BkstTsit of yeast 1 g / liter Sodium prcpcinate 0.5 g / liter Distilled water to a volume of 1 liter Adjust the pH value of the resulting solution to 71.0 with 1 N hydroxide solution sodium ', after which all was revealed in at 121 ° C for 20 minutes.

The cultures are made to a density .10 8 cells Ani, cells are separated by separation centrifuging, and then they are washed with an equal amount of the amount of an exhausted 0.1 molar cyan solution sodium trinate with a pH value of 5.6. After the transfer washing the cell is suspended in the same loop the amount of the trate buffer from the slurry of a sample is taken to determine the titer (count not cells), after the enzyme nitrosoguanides are added in an amount sufficient to obtain her death a total concentration of 50 µg / ml.

The cell suspension is incubated at temperature 37 ° C in a water bath, within 30 minutes, after now takes a sample to determine the titer, and the remainder spins and the separated cells are discharged washes equal to the volume of extracted 0.1 mol wego- potassium phosphate solution with the value of pH 7.0. Then the cells suspend again in an exhausted medium with minimal content salt, carbon-free, as follows with this composition: NH4NO3 1.0 g / liter K2HSPO4 0.25 g / iitT MgSC4-7 HgO 0.25 g / liter NaCl 0.005 g / liter FeSC4.7 H ^ O 0.001 g / liter Distilled water to a volume of 1 liter The pH of the slurry is then adjusted to its value at 7.0 with 1 N hydrochloric acid, then it comes out at 121 ° C on time minutes and poured the slurry on the plates in order selecting mutant cells 106 350 8 b) Selection and isolation of the Mycobacterium mutant fontuttum NIRRL B-8126.

Cells dormant as described above diluted it hides and sows on the fluids containing halves nutrient the following composition: (modified broth according to Firaser and Jerry 1968, J. Bioi.

OheaiL 205, 291—005): glycerin 10.0 g / liitor Per "HPC4" 8.4 g / L filter KHiP04 4.5 g / liter NH 2 Cl 2.0 g / liter NfeS047 H * Q 0.3 g / Mr FeGU.6Hfi 0.05 g / »r Distilled water to a volume of 1 liter.

Agar at a rate of 15 g / filter is added to the mixture and everything is revealed in the autoclave in the tamipera- tour 1S1QC for 30 minutes, then poured out on the uncut Petri dishes.

Breeding on such medium eliminates most of the nutritional auxotrols produced in the pro- during the mutagenesis process, for example, hoop is eliminated things that require vitamins, factors will increase stu and others, for the purpose of breeding W po a defined chemical medium.

After incubation at temperature 28 ° C within 7 days, the obtained colonies are transferred on the test plates suitable for the purpose of the series the lessons of the Irmitans, then transferred again on the control plates containing the preparation medium based on glycerin. Test plates is prepared by the method described by Pe- Teirson G, N, H. L. Lewis, and J. R. Davis in 1962 of the year in 'Prepairation o £ uniform disgpeirsione of cholesterol and other water -. carbcn insolufols scurces in agar modia "J. Lipid Researcn 3, 275-276.

On plates are used; a petition for minimal low salt content, such as that described above in point a) of example I. He adds to this statement agair in the amount of 15 g / filter and the corresponding source carbon, such as sytosterol or androstenedione (AD) at 1.0 g / liter. Then the obtained mixture Schweng is revealed in the autoclave within 30 minutes at temperature. 121 ° C. Exhausted, him All mixing is poured into a sterile mixer ka, mixes for a few minutes and then off left on the brightened Petri dishes. In the descriptions In this method, a certain difficulty is a tendency foaming medium that can be reduced sip, stirring the hot mixture and heating flames e-m surfaces of molten agar on plyttce. In this way, a homogeneous composition is obtained a mixture of water-insoluble sources of water carbon, which facilitates the production of very homogeneous different but opalescent agar plates.

Colonies that grew on the control plates but not on test plates containing AD as the only source of carbon, it cleans itself with the help of the entire line sowing on the culture plates agar. After the cultivation at the temperature of nature of 28 ° G, individual clones are selected from plates with agar medium diluted spatula dentist and re-tests with for the imitation of the colored plates contain using AD as a carbon source * Purified isolates, which still show the specific phonotype of the breeding knife parental flask is assessed in three-hole out. c) Assessment of breeding in a trajectory flask Fill three-barrel flasks with a capacity of 500 minutes is consumed with 100 ml portions of nutrient medium to be transferred carrying out biological transformation, fail the following ingredients: glycerin 10.0 g / liter NaaHPC4 8.4 g / L filter KHyP04 4.5 g / liter NH 2 2.0 g / liter MgSO4.7 HjO 0.3 g / Jat Fe018.6 HjO 0.05 g / liter Distilled water to 1 liter capacity The soybean flour is mixed with the nutrient 1 g / liter, then mixed with sftosterol in the amount of 10 g / liter. Then the koH would reveal herself in an autoclave for 20 manuA in teimiperattuirze 121 ° C, cooled to 28 ° C and inoculated ml of seed culture obtained as follows co: Purified isolates obtained as described in Fig zej is grown on agar slants at the temperature of 28 ° C. The mesh of cells with the skin is taken su, and is used for inoculating flasks with a capacity of 500 ml, containing the exhausted nutrient to a seed culture with the following composition: Broth (Difico) 8 g / liter Esktinaite from the root 1 g ^ lffltr Glicaryma 5 g / iMlfer Distilled water to a volume of 1 liter The pH value of the mixture was adjusted to 7.0 with 1 N sodium hydroxide solution and the mixture is released in an autoclave5 at the same time temperature of 121 ° C for 20 minutes. Vaccinated the flasks are incubated at 28 ° C body temperature g 712 hours.

As described above, 10 m for a seed culture 40 apply to inoculation of canned flasks by half The volume is 500 ml containing 1 (HT ml can be cured medium used to carry out the transfer education. These flasks are irffeubable in teirupeTatiu- temperature of 28-30 ° C and is shaken on a rotary 45 inputs, simultaneously downloading in different octes culture sample time, each with a capacity of 10 ml.

Plrc-these blocks are extracted by shaking them three times not with cMóiTkaetm methylation.

The portions of the extracts are tested using the chroma method. 50 togiriaidd of the streak with the use of sticky gel the miton and the solvent system are described previously, i.e., acetate mixtures ethyl and cyclohexane in the ratio by volume. 2: 3, and the method of choromaitograiii gas-liquid- 55 in. Confirmation of the presence of compounds of the formula 1, 2, 3 and 4 confirm the selective screen distribution steirol was carried out by the new mutamt made from the parents of my strain Mycobacte rium forttuirtuip, ATOC 8842.

Example of IL sitosterol filtration in compounds with formulas 1, 2, 3 and 4.

The medium used is the same as in the case of I place I in the evaluation section. breeding in ^ three-mouth flasksL revealed this poison 60 106 350 1 # at 12l ° C in 30 minutes, for the stejpnde is cooled to 30 ° C and then 10 parts are inoculated. seed culture Myicobactenium fortuiitum NRfRiL B-8128 mutant, which is obtained by the method described in the ointment day I. After inoculation to facilitate ho¬ in the soil of the soil, the mixture is incubated at temperature of 30 ° C within 336 hours of operation at the same time stirring. After incubation caJos6 is extracted with methylene chloride.

The extract drips through the diatomaceous earth layer entrance, and the passage evaporates to the socha under reduced pressure.

The remainder dissolves in a 10% solution chdorc & rmu in Skellysolve B and chroma- togcrafii on kiizemdlinking gel, using ja¬ for the eluent of Sk HysoLve B and its mixtures with vinegar ethide with increasing amounts of Ethics Acetate.

These mixtures elute compounds of formula I, 2 and 3. The compound of formula 4 flows out of the column % methanol in ethyl acetate. Relationship ki eLuiuije in the following order: compound of formula 1 -> • compound of formula 2 -> compound of formula 3 -> compound of formula 4. Descended the bundles show the following values of Rf na thin film chtromatograms (cykiothek- sain - ethyl acetate in the ratio 3: 2): compound of formula 1 R £ = 0 ^ 7) compound of formula II! Rf = 0.28 compound of formula 3 Rf = 04 * 6 compound of formula 4 Rf = 0.05j The composition of the respective fractions is determined by the method thin layer chromatography, joins firak-. similar composition - evaporates to dryness, receiving compounds of the formula with good yield accounts 1, 2, 3 and 4, After recrystallization from ethyl acetate, there is a compound of formula 1 which is a mixture two epimers in a 1: 1 ratio at temperature mp 100M112 ° C; the compound of formula II in the in oil form, the compound of formula III at temperature mp 122-124 ° C and the compound of formula 4 melting point 148M150 ° C.

Example IIL By following the example day II and using cholesterol instead of sitosterol, you get compounds of formulas 1, 2, 3 and 4.

Example IV. By following the example I go and use stigimasterol instead of sdtosteroHu, you get compounds of formulas 1, 2, 3 and 4.

Example V. By following the example II and using campesterol instead of sitosterol, you get compounds - with formulas I, 2, 3 and 4. * Example VI. By following the example II and using a mixture of aito- steroids with any of the steroids mentioned in the examples H <-V, the result is ki of formulas 1, 2, 3 and 4 ..

Example VII. Following the example of I. using instead of Mycobacteriuim fortuiltum ATOC 6642 sterile degradable microorganism role of the chosen way (genera: Arthrobacter, Ba- ciflUus, Brevibaeteri) Uin% Corynobacteriuim, Nocar- dia, ProtamSnobaoter, Serratia and Streptomycen, mutants of these microorganisms are obtained, oha- rakiteryzujaceujace to selective roo- a clade of steroids, possibly containing v molecule at position 17 aUriflical side chain at 2 — to 0 carbon atoms that are capable of growing the finishes of the formula 1, 2, 3 and 4 in the edge fermentation bowl.

(Example VIII. Following} as in the example II1-VI and using instead of the Mycobacterium mutant forfcuiitum NIRRL B-6108 the corresponding mutant obtained pissed as described in the example VH, you get compounds of formulas 1, 2, 3 and 4j Example IX. By following the example day I and using instead of the Mycobacterium strain fortuiltum ATCOC 6042 decomposing microorganism sterols selected from: Mycofcactertuum phlei, Mycobacterium smegmatisy Mycobacterium rhodo- ohrous, Mycobacterium imucosum and Mycobacterium butyricum, a mutant is obtained that has a character is characterized by the ability to selectively decompose possibly containing steroids ce in position 17 - alkali side chain o 2-40 carbon atoms and accumulative compounds of the formulas 1, 2, 3 and 4 in the broth Fer mentoring.

Example! X. By following the example roof IF-VI and using instead of the mutant Mycoba¬ cterium fortufttum NRRL 6-8128 suitable for mu the tant obtained as described in example IX, getting a compound of formulas 1, 2, 3 and 4. ao Example XI. Moving on like a glue day and using such a connection instead of sitosterol zec, such as androsteiKH4-dione4347, androstadienoyl, 4 Hdkm-3.17, dehydroepiandro sterone or testosterone, you get compounds of formulas 1, 2, 3 and i Example XII. By following the example day II and using instead of sitosterBu two or more more compounds such as sitosterol, cholesterol, sti <- gmaeterol, androstene-44dioneH3, (r7, androstadiene- -.l, 4-dione-13J17, dehydroeptiandrosterone and testosterone, 46 you get compounds of formulas 1, 2, 3 and 4.

Examples XXII. By following the example day XI and XII and using instead of the mutant Myco- baeteriium fortuituim NRRL B-8128 suitable mutant, obtained as described in example VII, 45 you get compounds of formulas 1, 2, 3 and 1 Example XIV. By following the example day XI and XXI, using instead of the mutant Mycoba¬ cterium fortuiitum B ^ 8128 a suitable mutant, obtained as described in Example IX was obtained they are compounds of the formulas 1, 2, 3 and 4j

Claims (12)

Patent claims Process for the preparation of 3a-H-7ap-Hmethate-4-hexahydroindanone-4 derivatives of the formulas 1, 2, 3 and 4, characterized in that Mycobacterium fortuitum NIRRL B-8128 is grown in an aqueous medium under aerobic conditions in the presence of a steroid optionally having a 17-position alkyl side chain of 2 to 10 carbon atoms, and isolating the compounds formed from the medium. 2. The method according to claim 1, characterized in that the steroid used is sitosterol, dholesterod, g5 stigmasterol, campesterol, androstene-4-dione-3.17, 60 106 350 11 12 andrastaddeno- hrb testosterone * 3. The method for the production of Za - ^ - ^^^ Hmcftylosescdowodoroil ^^ derivatives of the formulas 1, 2, 3 and 4, characterized by the cultivation of Mycofoactery fortuitum NRRL B-8128 in water medium under aerobic conditions, in the presence of the mixture two or more steroids, and the compounds are isolated from the nutrient medium. 4. As according to, reserve. 3. A method according to claim 3, characterized in that s / tosterol, cibdejsteapol, stigaBasterol, camtpesterol, andlroetenc - 4-) dioin, t3i, 17-, amdrostadienp ^ 1A e ^ hydro ^ piapd ^^ tercHn or testosterone, - 5. The method of producing 3cnH-7ia derivatives (1- - ^ ethylsize & cicwade ^ oWK ^ & formulas 1, 2, 3 and ¦ 4 ,: characterized by: & »that a mistand is grown; such a microorganism as Arthcobaeltar, BaciiBuis, Rrevibacteóum, Corynekacl ^ uim, Nbcardia, Pratairiiinoba-cteir ^ SerratLa or Streptomyces, characterized by the ability to selectively decompose steroids, possibly containing 17 alkyl side chains in the carriage and 2-10 carbon atoms and for the fermentation of the broth 3 -4 / nhydro »k3yprc ^ yl / -7apHmeityIosze of hydrogenindanedione-1,5, hemiacetal 3aa-H-4a- (3'-. -nydroxyproipyio) - ^ - nydroxy-7a (1-methyl-hexahydroxy-7a -4 boik: sye * tylo / -6a-thydroxy-7ap-methylxescdchydroxy-inclanon-1 oras 3aa ^ -to- / 3 '^ ydrc> xypropyl / - - ^ 5a-hydroxy-7a ^ HmeAydo (six) in the feed the cultures are carried out in an aqueous medium in variators of a grid, possibly containing a 17-position alkyl side chain with 12 carbon atoms, and the isolation of s Not the liquors produced from the nutrient *, ¦ 6.. 6. Method according to used. 5. The method of claim 5, wherein the mutant of the microorganism grows in an aqueous medium under aerobic conditions in the presence of a mixture of two leaves and more steroids. 7. Way according to zasitcrz. The method of claim 5, characterized in that the steroid is sitoslterol, holesterol, stigmasterol, kairrupesterol !, androstene-dione-S1, androstadien-yl, 4-dione-i3,17, denyKJroepiandrositron or testosterone. 8. Do according to the reservations. 6. A method according to claim 6, characterized in that steroids such as isitosterol, cholesterol, stigma-sterol, camipesterol, and; rostene-4-dione ^ 3, (17 <, androstationien - J, 4) are used as the ingredients of the steroid mixture. -dioin-3.17l, foam-denaturebrosteron and teststterone 9. Method of producing ScHH ^ afJ- derivatives 10.-methylsize hydroiiindanine of the formulas 1, 2, 3 and 4, characterized in that a mutant Mycobacterium is grown, which is characterized by the ability to selectively break down steroids, possibly containing an alkyl chain at position 17 We use 2— / 10 carbon atoms and to collect in the fermentation broth the 3aa-H- -Aa- (3'-ihydroixipropyl) -7a {1-methyl hexahydroindanedione-1.5, hemiacetal 3aa-H-4a / 3'-hydroxypropyl / -5a-'hydrolxy-7> apnmethyl hexahydro-20 indanone-d, S ^ laictoin 3aaJH-4a- / 2 '- (carboxylethyl) - ^ - liydroixy-7ap-methyl' ! anodo-Toalndanone> - -4 and 3a-H-4a ^ (3'-1hyoro-xy1proipyl) ^ 5a-hydroxy-7a | p-methyl-hexahydroindanone-1, and the cultures are carried out in a water medium 2s under aerobic conditions, The ¬ roid, possibly having a 17-position alkyl side chain with 2 to <10 carbon atoms, and is isolated from the nutrients produced by the compounds... 10. A method according to claim 9, characterized in that m that it cultivates and imutamates the microorganism in an aqueous medium under oxygen conditions in the presence of a mixture of two or more stereoids. 11. The method according to p. A method according to claim 9, characterized in that the steroid used is sitosterol, cholesterol, stigmasterol, campesteirol, amd'rosteine-4-dioin-3.17, amdrstad (ieriO- or testosterone. 12. The method according to claim The method of claim 10, characterized in that steroids such as sieve & terol, cholesterol, stigma-sterol, campesterol, amdro6teno-4-dicin-3.17, androstiadieinoHl, 4-d, ioin-3yl7, dehydroepiand / are used as constituents of the steroid mtiestzaininin. rosterone and testosterone 106 350 "6" formula HO Second3 HO 'Ka5r4